SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides forin vitroapplications

Abstract

Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Broadening the scope of sortagging

“…This novel approach for protein engineering was extensively expanded in the following years, e.g. for the ligation of uorescent proteins to a protein of interest, [36][37][38] to develop immunoassays with the aim of monitoring glucose levels, 39 to crosslink enzymes, 40 to generate fusion proteins that are inaccessible via direct expression, 41 to form an enzyme cascade within E. coli 42 and to ligate two domains of a protein. 43,44 Other studies focused on the ligation of two peptides [45][46][47] and of a peptide to a protein, [48][49][50][51][52][53] among others to demonstrate the purication of recombinant fusion proteins, 54 to functionalize amyloid-based polymers, 55 to assemble semisynthetic proteins or protein mimics 56,57 and to incorporate functional proteins into hydrogels.…”